The effects of DNA-reactive drugs on DNA in mammalian cells is being characterized and related to cytotoxic mechanisms. Alkaline elution methods are being utilized to measure DNA strand breaks, DNA-protein crosslinks, interstrand crosslinks and alkali-labile sites. The production of interstrand crosslinks by chloroethylnitrosoureas in human tumor cells was found to depend on a deficiency in certain cells in the capacity for DNA methylation repair. This deficiency enhanced the cytotoxic sensitivity of these cells. Mouse leukemia L1210 lines sensitive or resistant to cis-Pt (II) or melphalan were compared. Sensitivity correlated with the extent of interstrand crosslinking by these drugs. Sensitivity in mice correlated with crosslinking in vitro. The DNA intercalating agents, m-AMSA and mitoxanthrone, were found to produce protein-associated DNA strand breaks, but the extent of this effect produced by the 2 drugs did not correlate with cytotoxicity.